Methods of detecting modification of genetic material and monitoring processes thereof

ABSTRACT

The invention relates to a method for determining the activity of an enzyme or enediyne capable of altering the structure of a “substrate” nucleic acid from a first to a second state wherein the activity of the enzyme or enedyine is monitored using a chemiluminescent label that is either attached to the “substrate” nucleic acid or an oligonucleotide which is complementary thereto or the enzyme or enediyne product thereof.

FIELD OF THE INVENTION

This invention relates to a method of detecting and/or quantifying theactivity of enzymes involved in the modification of genetic material.The method is based on the use of labelled nucleic acids, wherein thelabels used may be, for example, fluorescent or chemiluminescentmolecules and the chemical properties of said labels may be modifieddepending upon the state of the nucleic acid in which the label issituated, either ab initio or as a result of a hybridisation step. Theinvention also extends to the use of the method in screening forpharmacological agents; agents identified thereby; and synthetic nucleicacid enzyme substrates.

BACKGROUND OF THE INVENTION

The replication, recombination, repair and other modification ofdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules allinvolve changes in the structure of genetic material and are offundamental importance to all living organisms. Examples of suchprocesses are enzymatic reactions where the enzymes are ligase,nuclease, integrase, transposase, helicase, gyrase, polymerase, primase,reverse transcriptase and. For example, DNA ligases are enzymes involvedin the modification of nucleic acid in organisms and can be divided intotwo classes, (i) the eukaryotic and viral enzymes which are ATPdependent, and (ii) the prokaryotic DNA ligases, which are dependent onNADH. In addition, prokaryotic ligases are unable to ligate blunt endedfragments and these distinct features of the prokaryotic enzymes makethem an attractive target for selective antibiosis. Work on eukaryoticsystems has also indicated that lack of ligase activity in humanscorrelates with certain pathological conditions.

The importance of assessing the activity of these enzymes has thereforeled to attempts to develop assay systems for the detection of factorsaffecting nucleic acid. Of particular interest is the ability to monitorbacterial or viral enzyme activity in the screening of novel compoundseither singly or in combination for anti-bacterial or anti-viralproperties due to their ability to inhibit the enzyme.

DESCRIPTION OF THE PRIOR ART

Current assays for assessing the activity of these enzymes, for exampleligase (FIG. 1 of the accompanying drawings) involve measurement ofenzyme intermediates or structural characterisation of the substrateand/or product. The majority of these assays also employ radioactivity,necessitating additional experimental precautions and incurring costsfor waste disposal. Recent assays have attempted to replace the use ofradioactivity with fluorescent labels for enzyme substrates.Alternatively, biological assays for DNA ligase activity have beendeveloped but are time consuming (at least 2 days), laborious andqualitative rather than quantitative. A more rapid biological assay hasbeen described (U.S. Pat. No. 5,976,806) but this involves the use ofcoupled transcription-translation systems with expression of a reportergene product (e.g. luciferase) in addition to the DNA ligase, makingthis multi-enzyme/multi-stage assay unsuitable for the high-throughputscreening of potential pharmaceutical compounds.

Assays for helicase (FIG. 2 of the accompanying drawings) have beendescribed which exploit the unwinding of double stranded nucleic acid.In one example (U.S. Pat. No. 5,958,696), a solid-phase derivative of adouble-stranded nucleic acid is prepared in which one of the strands islabelled with a radioisotope. Helicase activity is detected by itsability to release the labelled strand into the solution phase which canbe separated and measured. In a further example use is made of theability of certain markers to associate preferentially withmulti-stranded, e.g. double stranded nucleic acid, as opposed to singlestranded nucleic acid. Thus the marker will not associate with materialwhich is unwound by helicase activity.

The use of direct chemiluminescent-labelled oligonucleotide probes hasbeen described for the quantification of RNA in infectious organisms(U.S. Pat. No. 5,283,174, U.S. Pat. No. 5,399,491). In particular, thesemethods have shown utility in the detection of target nucleic acidsequences since use is made of the fact that certain molecules areprotected from degradation when associated with a nucleic acid duplexsuch that they retain an identifiable property as compared with theirdegraded counterpart. In a particular example, achemiluminescent-labelled oligonucleotide is exposed to chemicalconditions which bring about hydrolysis of the chemiluminescent moleculeand thus loss of chemiluminescence. If however duplex formation(hybridisation) with a complementary sequence has occurred thenchemiluminescence is retained subsequent to attempted hydrolysis due tothe protection imparted by the environment of the duplex. The sameprinciples can be applied to fluorescent molecules since it is knownthat structural modification can alter fluorescent properties as in thecleavage of the carbohydrate residue from4-methylumbelliferyl-β-D-galactose (Ishikawa and Kato, Scand J Immunol 8(Suppl. 7)1978).

SUMMARY OF THE INVENTION

In contrast to the prior art, we have developed systems which arecapable of discriminating between the nucleic acids that constitute thesubstrate of a reaction, and the product molecules that are formed as aresult of the action of enzymes acting on the said nucleic acids. Insuch actions the enzymes will cause a change of state in the substrate.

Preferred embodiments of the invention are designed to be extremelysensitive to apparently minor structural changes in the substrate. Forexample preferred embodiments are capable of detecting a “nick” in anucleic acid even where there are no bases missing in the nucleic acid.It will however be appreciated that the generation of or failure torepair such a nick may have major consequences in the replication,recombination and repair etc. of DNA and RNA. Likewise the preferredembodiments provide the ability to detect insertion, deletion,transposition of one or more bases or sequences in DNA or RNA as well aschanges in the non-covalent structure thereof.

Accordingly, in one aspect, this invention provides a method fordetermining the activity of a substance capable of altering thestructure of a nucleic acid from a first state to a second state, whichcomprises the steps of:

-   -   (a) providing in a test sample;        -   (i) said substance,        -   (ii) said nucleic acid; and optionally        -   (iii) one or more oligonucleotides complementary, at least            in part, to said nucleic acid when in said first or second            state wherein;            either, or both, of said oligonucleotide or nucleic acid            have associated therewith a label capable of providing an            output signal, and further wherein the stability of said            label against degradation is different depending upon            whether said nucleic acid is in said first or second state;    -   (b) exposing said test sample to degradation conditions;    -   (c) detecting said output signal and thereby determining whether        said nucleic acid is, at least predominantly, in said first or        second state; and thereby    -   (d) determining the activity of said substance.

The above method essentially detects a change in state of a nucleic acidcaused by substance activity.

Reference herein to the term activity includes reference to increased,decreased or zero activity of said substance.

Embodiments of the invention provide an efficient and reliable means ofmeasuring the activity or inhibition of activity of substances involvedin nucleic acid metabolism, and particularly in the repair andreplication of genetic material. In preferred embodiments themethodology uses labelled oligonucleotide sequences.

In some embodiments the labelled oligonucleotide sequences differentiatebetween the first and second state nucleic acid molecules appropriate tothese substances (i.e. between the two states of the nucleic acid) byselective binding (hybridisation) to the product (second state) moleculewhich in turn affects the chemical properties of the luminescentmolecule. In other embodiments the labelled oligonucleotide sequencesmay selectively hybridise to the unmodified (first state) molecule. Inyet other embodiments, the labelled oligonucleotide sequences arepre-prepared as a contrived nucleic acid where the labelledoligonucleotide can be thought of as already present in the nucleic acidmolecule.

The definition of nucleic acid as used in the present invention includesDNA, RNA, cDNA, gDNA, mRNA, tRNA, multi-stranded DNA, for example doubleor triple stranded DNA, as well as mixtures of such nucleic acids. Theterm nucleic acid used herein also encompasses strands of DNA or shortsequences or even a collection of unligated individual nucleic acidbases.

The “State” of a nucleic acid and its change from one state to the otherrefers to characteristics such as for example only whether a strandthereof is either intact or nicked; whether selected bases or sequencesthereof have been transposed in one or more strands; whether the duplexhas been unwound; whether the duplex has been cleaved; whether thenon-covalent structure has changed; whether strands thereof have beenintegrated; whether strands thereof have been ligated; whether thecollection of relevant bases has been assembled into a sequence. Forconvenience herein, the state of a nucleic acid is not deemed to changeagain once it has been subjected to substance activity. Thus if a nickedduplex is repaired by a ligase enzyme the duplex is said to have changedfrom a first (nicked) state to a second (repaired) state. If in a methoddescribed herein the two strands of repaired duplex are subsequentlyseparated, they are still regarded as being in the second state.

The term “hybridise” means the formation of a stable duplex or othermultiple-stranded molecule between complementary single strandedmolecules.

Embodiments of the invention provide simple, rapid and robust assays tomeasure the activity of substances having an affect on nucleic acidmetabolism. Whilst useful in many situations where the assessment ofsuch activity is required, these assays are particularly suitable forthe screening of putative anti-bacterial and anti-viral compoundscapable of inhibiting the said substance activity.

Where substance activity is assessed, said substance may be one or moreof ligase, nuclease, transposase, integrase, primase, helicase, gyrase,polymerase, reverse transcriptase, a topoisomerase or an enediyne.

Enediynes are naturally occurring organic molecules (for e.g.calicheamicin and esperamicin) that behave as restriction endonucleasesas they have the ability to cleave duplex nucleic acid and it is thisability to convert a nucleic acid molecule from a first to a secondstate that enables these molecules to be included within the scope ofthis invention. Whilst their action is non-catalytic they have a 1:1reaction with a nucleic acid molecule. The enediyne class of moleculeshave been described in detail in the following publication (Borders D B& Doyle T W 1995 ‘Enediyne Antibiotics as Anti-Tumour Agents’ (Dekker,New York)) and their ability to mimic the activity of restrictionendonucleases is described in the following paper: Biggins et al PNAS 9713537-13542.

It therefore follows that enediynes are substances falling within thescope of the invention since they are able to convert a nucleic acidmolecule from a first to a second state and thus, using the technologydescribed herein, the activity of these molecules can be assayed.Furthermore, using the invention described herein the presence of thesemolecules, and thus the presence of their activity within a sample, canalso be identified. Furthermore, given the ability of these molecules toalter the molecular structure of a nucleic acid from a first to a secondstate it also follows that, using the invention described herein, it ispossible to screen for molecules that regulate the activity of enediynesand so identify molecules or agents which are active pharmacologicallyas agonists or antagonists thereof.

In a particular embodiment for monitoring the activity of ligase, saidnucleic acid is multi-stranded and step (a) involves exposure of adouble-stranded nucleic acid to ligase and after exposure to the ligase,the sample is subjected to a raised temperature to cause any unligatednucleic acid, at least partially, to yield single strands.

The use of temperature control selectively to melt or selectively tore-hybridise unligated nucleic acid fragments provides an opportune wayof differentiating between ligated and unligated nucleic acid. In onetechnique, the raised temperature is controlled so that unligatednucleic acid at least partially separates but ligated nucleic acid doesnot.

As noted above the invention may be used for monitoring a wide range ofdifferent enzymes. Thus in another embodiment, for monitoring helicaseactivity, the nucleic acid is multi-stranded and step (a) involvesexposing the nucleic acid to helicase in an environment which allows atleast partial unwinding of the nucleic acid.

In a preferred methodology for determining the activity of helicase saidoligonucleotide, referred to herein above, is omitted from the testsample and, instead, said nucleic acid is provided with said label.

Where the nucleic acid is multi-stranded and the enzyme is a helicasethe nucleic acid is changed between first and second states, by:

-   -   (i) separating at least a portion of one of the strands of the        nucleic acid from another thereof to provide a single strand        portion; and optionally,    -   (ii) contacting the sample with one of said oligonucleotides,        wherein one of said oligonucleotides or said nucleic acid has        associated therewith said label, and said oligonucleotide is        capable of hybridising to said single strand portion of the        nucleic acid.

In yet another embodiment, for monitoring the activity of a polymeraseor primase, the nucleic acid is in the form of nucleotides or shortfragments thereof and part (a) involves exposure of said nucleic acid toa polymerase in an environment which allows said bases and/or nucleicacid strands to join.

It will be appreciated that the assay methods may be designed to provideone of two different endpoints; in one, the label of the labellednucleic acid is relatively affected if said nucleic acid has undergone achange in state; in the other, the label of the labelled nucleic acid isrelatively unaffected if said nucleic acid has undergone a change instate.

Where said enzyme when active acts to repair at least one of a nick orother discontinuity in an interrupted strand of a multi-stranded nucleicacid, to provide a repaired strand, part (a) may comprise the steps of:

-   -   (i) raising the temperature of the sample to a temperature in        excess of the temperature required to cause the interrupted        strand to separate from the or each remaining strand,        (irrespective of whether the interrupted strand has been        repaired);    -   (ii) contacting the sample with said labelled oligonucleotide,        said labelled oligonucleotide being capable of hybridising to        the repaired strand;    -   (iii) reducing the temperature of the sample to a temperature        below the melting point of a duplex containing the repaired        strand, but above the melting point of a duplex containing the        non-repaired portions of said interrupted strand, thereby to        allow said labelled oligonucleotide to hybridise to said        repaired interrupted strand if present; and    -   (b) thereafter exposing said sample to said degradation        conditions and subsequently detecting the activity of the label,    -   whereby in the said detection step, the presence or amount of        relatively unaffected label indicates the presence or amount of        activity respectively of said repair enzyme.

In this arrangement, hybridisation of the labelled oligonucleotide tothe repaired strand when present, results in a complex in which thelabel is relatively protected against degradation.

Of course a similar method may be used where, instead of repairing anick or discontinuity, an enzyme when active generates at least one of anick or other discontinuity by inter-base cleavage in at least onetarget strand of a multi-stranded nucleic acid to create an interruptedtarget strand. In this instance, part

-   (a) may comprise:—    -   (i) raising the temperature of the sample to a temperature in        excess of the temperature required to cause the target strand of        the nucleic acid to separate from the or each remaining strand        (irrespective of whether the enzyme has been active to create an        interrupted target strand);    -   (ii) contacting the sample with said labelled oligonucleotide,        said labelled oligonucleotide being capable of hybridising to        said target strand when in uninterrupted form;    -   (iii) reducing the temperature of the sample to a temperature        below that at which said uninterrupted target strand will        hydridise to the labelled oligonucleotide, but above the        temperature at which the separated interrupted portions of the        target strand can hybridise to said remainder of the nucleic        acid, thereby to allow said labelled oligonucleotide to        hybridise to said uninterrupted target strand if present, and;    -   (b) thereafter exposing said sample to said degradation        conditions and subsequently detecting the activity of the label,    -   whereby in said detection step, the presence or amount of        relatively affected label indicates the presence or amount        respectively of said enzyme capable of yielding a cleaved        molecule.

In this assay, if uninterrupted strands are present in the sample, thelabelled oligonucleotide will hybridise thereto in step (iii) to form acomplex in which the label is relatively protected.

In each of the above instances, the sample may be contacted with thelabelled oligonucleotide before or after the step of raising thetemperature (Step (i)).

In another aspect or embodiment of the invention, said labelled nucleicacid may comprise a complex made up of said nucleic acid and a label,said nucleic acid being capable of being acted upon by a substancewhereby, on said substance being active, the nucleic acid changes fromsaid first state to said second state, thereby changing the stability ofthe label. Such a complex is referred to elsewhere herein as a contrivedsubstrate.

In another embodiment, said nucleic acid is in the form of a collectionof free (i.e. unligated) nucleotides, and said enzyme is active to causeor allow selected free nucleotides to be joined to yield a second statein which they form at least one strand of a product nucleic acid, andpart (a) involves contacting said sample with a labelled oligonucleotidedesigned to hybridise with said product nucleic acid.

In another embodiment said substance is a nuclease or an enediyne, saidoligonucleotide is omitted from said sample and said nucleic acid, whichis multi-stranded and includes a cleavage point is provided with saidlabel and step (a) further includes subjecting said sample to atemperature that causes any cleaved nucleic acid to separate into singlestrands. Alternatively, this embodiment of the invention may be modifiedsuch that said oligonucleotide is not omitted from said sample and thussaid nucleic acid is not provided with said label. In this embodiment ofthe invention said nuclease or enediyne acts on said nucleic acid thuscleaving same so that, when said sample is subject to a temperature thatcauses any cleaved nucleic acid to separate into single strands saidlabelled oligonucleotide can bind to a selected one of said strands forthe purpose of carrying out the assay.

The detection of the output signal from the label assay may involve theuse of one or more of colourimetric, fluorimetric or chemiluminescentmeans.

The label may conveniently be a fluorescent or chemiluminescentmolecule, for example an acridinium salt.

As well as detecting substance activity, the methods disclosed hereinmay be used for screening for modulatory activity. Thus in anotheraspect this invention provides a method for screening an agent formodulatory activity in relation to a substance capable of altering thestructure of a nucleic acid from a first state to a second state, whichcomprises the steps of:

-   -   (a) providing in a test sample:        -   (i) said substance;        -   (ii) said nucleic acid;        -   (iii) an agent to be tested; and optioinally        -   (iv) at least one oligonucleotide complementary, at least in            part, to said nucleic acid, when in said first or second            state wherein;        -   either, or both, of said oligonucleotide and said nucleic            acid has associated therewith a label capable of providing            an output signal, and further wherein the stability of said            label against degradation is different depending on whether            said nucleic acid is in said first or second state;    -   (b) exposing said test sample to degradation conditions;    -   (c) detecting said output signal and thereby determining whether        said nucleic acid is, at least predominantly, in said first or        second state; and thereby    -   (d) determining the activity of said substance and thus the        modulatory activity of said agent.

The above method may be used to screen substances for pharmacologicalactivity.

The invention also extends to a nucleic acid for use in detecting theactivity of a predetermined substance, said nucleic acid being capableof reactivity with said substance and having an associated label, thelocation of the label and the configuration of the nucleic acid beingselected such that, in use, when said substance is active on saidnucleic acid it changes the state of the nucleic acid from a first stateto a second state, and wherein the stability of said label againstdegradation in a subsequent reaction is different according to whethersaid nucleic acid is in its first or second state.

The invention also extends to a method of detecting whether a nucleicacid in a sample has undergone an event resulting in said nucleic acidchanging from a first state to a second state,

As noted previously, the use of selective temperature managementprovides an important way of detecting whether an enzyme creates orrepairs a nick in a substrate nucleic acid.

In another aspect this invention provides a method for detecting in asample the activity or presence of an enzyme capable of repairing aninterrupted nucleic acid strand to form a repaired nucleic acid strand,which comprises the steps of:—

-   -   (a) providing in said sample;        -   (i) a multi-stranded nucleic acid having an interrupted            target strand made up of at least two interrupted portions            capable of being ligated by said enzyme when active;    -   (b) applying the sample to a temperature in excess of the        melting temperature of at least one of the interrupted portions        of the unrepaired interrupted target strand, but below the        melting temperature of the repaired interrupted strand, whereby        there is little or no hybridisation of at least one of the        unrepaired interrupted portions of the target strand to the        complementary strand or strands, and    -   (c) thereby determining at least one of the activity or presence        of said enzyme.

In another aspect this invention provides a method for detecting in asample the activity or presence of an enzyme capable of generating anick or other discontinuity in at least one target strand of amulti-stranded nucleic acid to create an interrupted target strand,which comprises the steps of:

-   -   (a) providing in said sample;        -   (i) a multi-stranded nucleic acid incorporating a site at            which a nick or discontinuity may be generated or created;    -   (b) applying the sample to a temperature in excess of the        melting temperature of at least one of the unligated portions of        the interrupted target strand (if present), whereby there is        little or no hybridisation of said at least one of the unligated        portions of the interrupted strand to the complementary strand        or strands and;    -   (c) thereby determining at least one of the activity or presence        of said enzyme.

In either of the above determining steps the methodology may includeintroducing into the sample a labelled oligonucleotide complementary toat least a portion of the said complementary strand of the nucleic acid,thereby to detect the presence or amount of hybridisation between therepaired or uninterrupted strand and said complementary strand.Alternatively said determining step may include introducing into thesample a labelled oligonucleotide complementary to one of the fragmentsof interrupted strand thereby to detect the presence or amount ofhybridisation.

In one arrangement said nucleic acid is selected with regard to theinterrupted fragments or the active site such that there are threedifferent melting temperatures as follows:

-   -   (i) a melting temperature of a first fragment of the interrupted        strand of the substrate;    -   (ii) a melting temperature of a second fragment of the        interrupted strand of the substrate;    -   (iii) a melting temperature of an uninterrupted strand of the        substrate.

It will be appreciated that the melting temperatures of the fragmentsand their lengths may be controlled in various ways, for example bytheir relative lengths, or by introducing selected mismatches in thesequences.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

In one embodiment, an oligonucleotide sequence is labelled with achemiluminescent molecule that can be rendered non-chemiluminescent bydissociation of one or more bonds but is protected from saiddissociation when the labelled oligonucleotide sequence constitutes partof a multi-stranded nucleic acid, for example a duplex. Surprisingly wehave found that, under the conditions used to bring about dissociationof the chemiluminescent molecule, a nucleic acid containing an unligatedstrand is incapable of offering protection against dissociation.

Thus in embodiments described below a labelled chemiluminescentoligonucleotide, is synthesised which is complementary to the sequenceof interest, the sequence of interest being the substrate or product ofthe enzyme or enediyne of interest. A solution of the labelledoligonucleotide is admixed with a solution of the said sequence ofinterest under conditions conducive to hybridisation. The reactionmixture is then exposed to chemical, enzymatic and/or physicaldegradation conditions known to bring about dissociation of thechemiluminescent molecule and thus render it non-chemiluminescent. Thereaction vessel is then placed in a luminometer and reagents added tobring about the chemiluminescent reaction whilst monitoring any emittedlight. Alternatively, if the kinetics of the chemiluminescent reactionare sufficiently slow, the chemiluminescence can be initiated prior toplacing the reaction vessel into the luminometer. The presence of thesequence of interest and so the formation of a duplex with the labelledoligonucleotide results in retention of chemiluminescence whereas theabsence of the sequence of interest and so the inability to form aduplex with the labelled oligonucleotide results in the loss ofchemiluminescence. Consequently, it is possible to determine therelative amounts of the sequence of interest.

One embodiment provides an assay for ligase or nuclease enzymes orenediynes, since the substrate and product molecules differ by beingligated or unligated sequences. Thus, for example, if “nicked” DNA isexposed to a preparation possessing ligase activity the formation ofligated product will be revealed by hybridisation to thechemiluminescence labelled oligonucleotide and retention ofchemiluminescence due to protection from, for example, conditionscapable of hydrolysing uncomplexed chemiluminescent label.

Preferably, the nucleic acid in a sample is exposed to ligase and afterexposure to the ligase, the sample is subjected to a raised temperatureto cause nucleic acid in the sample to denature or separate, andsubsequently the temperature is reduced to allow the nucleic acid torehybridise.

It is preferred that the raised temperature used is high enough suchthat unligated nucleic acid separates but ligated nucleic acid does not.Preferably, the temperature used is adjusted according to thestoichiometry of the hybridisation reaction.

A surprising finding is that in many cases the enzyme or enediyne iscapable of functioning even when the nucleic acid to be acted uponpossesses a label moiety. Thus a further aspect of the invention definedabove involves the use of a pre-formed, labelled enzyme or enediyne‘substrate’ (referred to as a contrived substrate) which comprises amulti-stranded e.g. a double-stranded oligonucleotide sequence whereinone of the strands possesses a hydrolysable chemiluminescent label asdescribed above.

Optionally, said other strand possesses a ‘nick’. Upon exposure toelevated temperature, for example, the unligated duplex is incapable ofprotecting the chemiluminescent label from hydrolysis whereas theligated duplex, formed as the result of prior ligase activity, protectsthe chemiluminescent label from hydrolysis.

The embodiments described herein disclose ways of assessing the activityof enzymes responsible for the interconversion of ligated and unligatedforms of genetic material which are potential targets for the screeningof putative pharmacologically active compounds.

Similarly, the same principles can be applied to the assay of thoseenzymes which catalyse the insertion (integrase) or transposition(transposase) of discrete nucleotide sequences within a given genesequence. Here use is made of an appropriate labelled oligonucleotidesequence which is capable of hybridising with the product sequence butnot the substrate sequence. In this way, not only can the activity ofintegrase or transposase preparations be assessed but it is possible todetermine whether chemical compounds added into the reaction mixture arecapable of inhibiting the enzyme activity and may thus have utility aspharmacological agents.

Enzymes of the class exemplified by nuclease, ligase, integrase andtransposase all have the common feature of catalysing the covalentmodification of genetic material.

There also exist enzymes which catalyse changes in secondary structureof the genetic material, such enzymes being exemplified by helicase.Activity of these enzymes results in the formation of sections of“unwound” nucleic acid. Here, use is made of the fact that the “unwound”product nucleic acid sequence produced as a result of the enzymeactivity is accessible to binding by a complementary labelledoligonucleotide sequence in contrast to the substrate duplex nucleicacid sequence.

As above it may be desired to use a pre-formed substrate includingdouble-stranded nucleic acid and already containing the luminescentlabelled oligonucleotide sequence and in which the luminescent label isprotected from degradation (e.g. hydrolysis) due to its position withinthe double stranded nucleic acid. The presence of helicase activity thencauses the duplex nucleic acid to be unwound hence exposing theluminescent label to hydrolysis. In this case, luminescence intensity isinversely proportional to helicase activity.

In a further preferred embodiment of the invention, the nucleic acid isexposed to helicase in an environment which allows unwinding of strandsmaking up the nucleic acid, and a material is included in the samplewhich could alter activity of the helicase, and then conditions areprovided for the strands to rehybridise in the presence of labelledoligonucleotides complementary to one strand of the above unwoundnucleic acid.

In certain situations, as a variation of the situation when labelledoligonucleotide sequence binding is used subsequent to performing theenzymic reaction, it may be appropriate to design the labelledoligonucleotide sequence to bind to the substrate rather than theproduct of the enzyme reaction.

The inventive principles herein can also be applied to those situationswhere a nucleic acid product is created from small precursors such asindividual bases since the product of the enzyme reaction is capable ofhybridisation with a labelled complementary oligonucleotide sequencewhereas the reactants are not. Examples of such enzymes are primase,polymerase and reverse transcriptase.

In another embodiment of the invention, nucleic acids/strands areexposed to polymerase in an environment which allows nucleicacids/strands to join, and included in the sample are one or morenucleic acids/strands which may be complementary to the joined nucleicacids/strands and providing conditions for said joined and complementarynucleic acids/strands to hybridise.

Normal enzyme activity gives rise to a nucleic acid capable ofhybridisation with a complementary labelled oligonucleotide sequence andthe subsequently formed duplex protects the label from degradation.Inhibition of the enzyme results in no duplex being formed and hence noprotection of the label from induced degradation. The subsequentmeasurement of luminescence of a marker such as a chemiluminescent orfluorescent label on the oligonucleotide is therefore a quantitativeindicator of the activity or otherwise of the enzyme concerned.

Further, luminescent labels also have the advantage that it is possibleto configure “multichannel” assays. There exist in the literaturereports of using both wavelength and temporal discrimination to enablemixtures of labels to be quantified simultaneously yet independently(U.S. Pat. No. 5,827,656). This same principle can be used to goodeffect in the present teachings where, for example, it may be desirableto screen chemical compounds simultaneously for inhibitory activitytoward for e.g. ligase and integrase. Based upon the disclosures herein,one skilled in the art will readily appreciate how suitable multichannelassays may be designed and used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the action of a ligase enzyme inwhich parts of a nucleic acid sequence are ligated;

FIG. 2 shows a schematic diagram of the action of a helicase enzyme inwhich the individual strands of a double-stranded nucleic acid are“unwrapped”;

FIG. 3 is a schematic diagram representing the steps involved in a firstembodiment of this invention to assay for ligase activity;

FIG. 4 is a schematic diagram representing the steps involved in asecond embodiment of this invention also to assay for ligase activity;

FIG. 5 is a schematic diagram representing the steps involved in a thirdembodiment of the invention whereby a contrived labelled substratenucleic acid is used to assay for ligase activity;

FIG. 6 is a schematic diagram representing the steps involved in afourth embodiment of the invention to assay for activity of enzymes suchas DNA helicase;

FIG. 7 is a schematic diagram representing the steps involved in a fifthembodiment of the invention to assay for enzymes such as RNA polymeraseactive on a multi-stranded DNA template;

FIG. 8 is a schematic diagram representing the steps involved in a sixthembodiment of the invention to assay for activity of enzymes such asreverse transcriptase or primase which act on a single-strandedtemplate;

FIG. 9 is a schematic diagram representing the steps involved in aseventh embodiment of the invention to assay for the activity ofenediynes or enzymes, such as nucleases, which are active onmulti-stranded DNA;

FIG. 10 is a schematic diagram representing the steps involved in aneighth embodiment of the invention to assay for the activity of enzymessuch as integrases which act on oligonucleotides;

FIG. 11 is a schematic diagram representing the steps involved in aninth embodiment of the invention to assay for the activity of enzymessuch as topoisomerases which act on double-stranded nucleic acidmolecules;

FIG. 12 shows the results of the experiment of Example 1 where EDTA isused as an inhibitor of the ligase enzyme;

FIG. 13 shows the results of the experiment of Example 2 where di-deoxythymidine triphosphate (ddTTP) is used as an inhibitor of the reversetranscriptase enzyme;

FIG. 14 shows the results of the experiment of Example 3 where theactivity of helicase is measured at 3 enzyme:substrate ratios;

FIG. 15 shows the results of Example 4 where EDTA is used as aninhibitor of the viral DNA dependent RNA polymerase enzyme; and

FIG. 16 shows the results of the experiment of Example 5 whererifampicin is used as an inhibitor of the bacterial DNA dependents RNApolymerase enzyme.

In various embodiments of this invention the change in state of asubstrate nucleic acid is detected by causing the formation of a complexmade up of one of the strands of the substrate nucleic acid (which mayor may not be the strand directly affected by the change in state) and alabelled oligonucleotide which is designed so that its protectionagainst degradation in a subsequent degradation step is differentaccording to whether the substrate nucleic acid is in its first orsecond state. Following exposure to a degradation step, the label signalis detected in a manner appropriate to the label being used, and fromthis may be determined the state of the substrate nucleic acid.

For a better understanding of the various techniques we now describe anumber of different schemes, with reference to the schematic diagrams inthe Figures.

Scheme A (FIG. 3)

A first oligonucleotide duplex is synthesised which comprises a firststrand 10 of nucleotides complementary to a second strand 12. When boundin a nucleic acid duplex with the first strand, the second strand canexist either as an intact (ligated) strand 12 _(L) or a “nicked”unligated strand 12 _(U). For the purposes of this scheme, which isdesigned to detect ligase activity, or factors influencing suchactivity, the nucleic acid duplex is synthesised with the second strand12 _(U) nicked or unligated. The unligated second strand 12 _(U)represents at least part of a strand capable of acting as a ligaseenzyme substrate which is converted to the ligated strand 12 _(L) by theaction of the enzyme. In this assay, the two states of the substratenucleic acid are the one in which the second strand is unligated, andthe one in which the strand is ligated (ii).

A third oligonucleotide 14 is synthesised which is identical to thefirst strand 10 (and thus complementary to the second strand 12), butwhich further comprises a “linker” moiety 16 to which can be attached achemiluminescent or fluorescent emitter molecule 18. In certainapplications it may not be necessary for the third oligonucleotide to beidentical to the first strand; base mismatches may be allowed providedthat the third oligonucleotide is capable of hybridising stably to thesecond strand.

Scheme A comprises the following stages, in which the bracketed romannumerals relate to the steps illustrated in FIG. 3.

-   -   (i) A reagent is provided consisting of duplex strands of the        first strand 10 hybridised to the second strand 12 _(U), the        second strand 12 _(U) being “nicked” or unligated.    -   (ii) The reagent of step (i) is exposed to a ligase enzyme with        or without inhibitors and co-factors. (The left hand side of the        Figure shows the condition where there is enzyme activity and        the right hand side shows the condition where there is no such        activity; this also applies in the remainder of FIGS. 3 to 8).    -   (iii) A labelled oligonucleotide 14 is introduced into the        sample, and the temperature of the sample is raised to cause the        first and second strands to separate.    -   (iv) The temperature is reduced to a temperature below the        hybridisation temperature of the ligated (intact) strands 12        _(L) but above the temperature of the unligated fragments of the        second strand 12 _(U), so that some of the second ligated        strands 12 _(L) will hybridise to the labelled oligonucleotide        14 instead of to the first strand 10. However, as the        temperature is above the hybridisation temperature of the        unligated short target strands, the fragments of the unligated        12 _(U) will not hybridise to the labelled oligonucleotide 14.    -   (v) The sample is then subjected to conditions which degrade the        label 18, e.g. by hydrolysis or dissociation of the label        (hereinafter referred to generally as degradation conditions).        The nature of the labelled oligonucleotide is such that, if the        labelled oligonucleotide has hybridised to an intact strand 12        _(L), the output signal from the label 18 will be substantially        unaffected. On the other hand, if the labelled oligonucleotide        has not hybridised (or has only partially hybridised), it will        not be protected against the degradation conditions and so the        light output signal will be affected (it may be non-existent or        it may be in an altered form).    -   (vi) The chemiluminescent reaction is initiated and the light        output is measured or fluorescence is measured depending on the        nature of the label.    -   (vii) The light output signal is proportional to ligase        activity.        Scheme B—FIG. 4

This scheme is similar to Scheme A in that it uses a first strand 10 anda second strand 12 which may be in ligated form (12 _(L)) or unligatedform (12 _(U)), and a labelled oligonucleotide is used. However in thisexample, the labelled oligonucleotide 14 is designed to hybridise withthe first strand 10 rather than the second strand.

-   -   (i) Substrate duplex strands made up of the first strand 10        hybridised to the second strand 12 _(U) in unligated form are        provided in the sample.    -   (ii) The sample is exposed to ligase with or without inhibitors,        co-factors etc.    -   (iii) The temperature of the sample is raised to a temperature        high enough to cause unligated second strands 12 _(U) to        separate from the first strand, but not high enough to cause        ligated second strands 12 _(L) to separate.    -   (iv) The sample is exposed to a labelled oligonucleotide 14        complementary to the first strand and hybridisation is allowed        to occur to any of the unhybridised first strands 10.    -   (v) The sample is subjected to degradation conditions such that        the chemiluminescent or fluorescent activity of any unhybridised        labelled oligonucleotide 14 is affected but that the activity of        any labelled oligonucleotide 14 hybridised to complementary        strand (in this instance the first strand 10) is substantially        unaffected.    -   (vi) The chemiluminescent reaction is initiated and the light        output is measured or fluorescence is measured depending on the        nature of the label.    -   (vii) The light output signal is inversely proportional to        ligase activity.        Scheme C—(FIG. 5)

In this scheme a contrived substrate nucleic acid duplex 20 isengineered.

-   -   (i) The substrate nucleic acid duplex 20 is made up of a first        strand 22 hybridised to a nicked or unligated second strand 24        _(U). A linker moiety 26 connects a label 28 to the first strand        22. The contrived substrate nucleic acid duplex 20 is designed        with the label 28 positioned relative to the nick in the        unligated strand 24 _(U) such that the label is relatively        unprotected against degradation conditions whilst the second        strand is unligated but is relatively protected against such        conditions if the second strand is ligated by enzyme activity.        Whilst in the schematic representation the label is shown        directly opposite the nick, the relative locations of the label        and the nick in the unligated strand may be varied and indeed        the nick may be several bases away from the location of the        label on the opposite strand. Suitable location of the nick        relative to the label and to the ends of the contrived substrate        may be determined empirically, based on the disclosures of U.S.        Pat. Nos. 5,283,174 and 5,399,491.

(ii) The contrived substrate 20 is exposed to ligase with or withoutinhibitors, co-factors etc. In the presence of ligase activity, theunligated second strand 24 _(U) is repaired to provide a ligated strand24 _(L). In the absence of enzyme activity the second strand 24 _(U) isunrepaired.

-   -   (iii) The sample is then raised to a temperature sufficiently        high to cause unligated second strands 24 _(U) to separate from        the first strand 22 but not high enough to cause ligated second        strands 24 _(L) to separate from the first strand 22. The sample        is then subjected to degradation conditions to degrade the        activity of the label 28 if the first strand is not protected by        the ligated second strand 24 _(L).    -   (iv) The chemiluminescent reaction is initiated and the light        output is measured or fluorescence is measured depending on the        nature of the label.    -   (v) The light signal output is proportional to ligase activity.        Scheme D—(FIG. 6)

This scheme is intended for monitoring for activity of an enzyme such asDNA helicase which causes separation of two strands.

-   -   (i) A contrived duplex strand 30 is provided with a first strand        32 having a label 34 attached by means of a linker moiety. The        first strand 32 is hybridised to a second strand 38.    -   (ii) The contrived substrate 30 is exposed to helicase with or        without inhibitors, co-factors etc. In the presence of active        helicase the first and second strands 32 and 38 are separated by        enzyme activity to change the state of the duplex but, in the        absence of such activity, the state of the contrived substrate        30 is unaltered.    -   (iii) The sample is exposed to degradation conditions to degrade        the activity of the chemiluminescent or fluorescent label 34. If        the first strand 32 has become separated from the second strand        38 then the activity of the label 34 will be compromised, but if        the enzyme is not active the label 34 will be relatively        protected.    -   (iv) The sample is subjected to conditions to cause loss of        chemiluminescent or fluorescent activity if unprotected.    -   (v) The light output signal is inversely proportional to        helicase activity.        Scheme E—(FIG. 7)

This Scheme is useful for monitoring activity of an enzyme such as RNApolymerase or other enzymes which assemble the ribo-nucleosidetriphosphate “building blocks” 40 into a nucleic acid sequence 42.

-   -   (i) A suitable duplex DNA or RNA template (not shown) is        provided in a sample together with ribo-nucleoside triphosphates        40, the enzyme being tested and any required co-factors or        inhibitors.    -   (ii) If the enzyme is uninhibited it assembles a single stranded        ribo-nucleotide product 42; otherwise the ribo-nucleoside        triphosphates 40 remain separate.    -   (iii) A labelled oligonucleotide 44 complementary to the product        of enzyme activity of (ii) is introduced into the sample.    -   (iv) The labelled oligonucleotide 44 hybridises to the assembled        product 42 if present.    -   (v) The sample is subjected to degradation conditions to cause        loss of chemiluminescent or fluorescent activity. Where the        labelled oligonucleotide 44 hybridises to the product 42 the        stability of the label 46 is relatively unaffected as compared        to where the labelled oligonucleotide has no assembled strand to        which to hybridise.    -   (vi) The chemiluminescent reaction is initiated and the light        output is measured or fluorescence is measured depending on the        nature of the label.    -   (vii) The light output signal is proportional to enzyme        activity.        Scheme F

This scheme is designed for monitoring activity etc. of enzymes such asreverse transcriptase or primase.

-   -   (i) A sample is made up comprising a single-stranded template 48        together with nucleoside triphosphates 50, the enzyme being        tested, and one or more co-factors or inhibitors if required.    -   (ii) Where active, the enzyme generates a complementary target        strand 52 on the template 48; where inactive no complementary        strand is generated.    -   (iii) A labelled oligonucleotide 54 complementary to the        enzyme-synthesised target strand 52 is introduced into the        sample.    -   (iv) The temperature is cycled to cause the template 48 and the        enzyme-synthesised target strand 52 to separate and then lowered        to allow hybridisation; if the target strand 52 is present, some        of these will hybridise to the labelled oligonucleotide 54.    -   (v) The sample is subjected to degradation conditions to cause        loss of chemiluminescence or fluorescent activity such that        unhybridised labelled oligonucleotide loses activity.    -   (vi) The chemiluminescent reaction is initiated and the light        output is measured or fluorescence is measured depending on the        nature of the label.    -   (vii) The light output signal from the label 50 is proportional        to enzyme activity.        Scheme G (FIG. 9)

(a) DNA duplex which comprises a site specific cleavage point is firstsynthesised. A chemiluminescent label (AE label) is then attached to oneof the strands of the duplex at a site sufficiently remote from the saidcleavage point so that the label will not interfere with the activity ofa nuclease enzyme. Most ideally, the said label is positioned so as toavoid any steric hindrance between itself and the nuclease enzyme. If acleavage agent or nuclease enzyme is not present the labelled duplexwill remain substantially intact (left-hand side of FIG. 9).Alternatively, if a cleavage agent or nuclease enzyme is present it willact upon the site specific cleavage point, cleaving the DNA. Thus, whenthe DNA is subsequently exposed to a suitably selected melt temperaturethe cleaved strand separates away from its complementary strand leavingthe chemiluminescent label exposed. Thereafter, when exposed tohydrolysing conditions the chemiluminescent label is destroyed. Incontrast, where the enzyme is absent, or inactive, cleavage of theduplex does not occur and thus the chemiluminescent label can shelterfrom the effects of hydrolysis within the duplex coil and so retain itsfunctionality. In this way, the output of the chemiluminescent signal isinversely proportional to cleavage activity. Thus as cleavage increases,due to the increased presence or activity of the nuclease enzyme, moreand more chemiluminescent label is destroyed and so the chemiluminescentsignal declines.

Scheme G also illustrates the steps involved in the enediyne cleavageassay. As above, in the presence of an enediyne the labelled duplex iscleaved and subsequently, upon exposure to melt conditions, if cleavagehas taken place a cleaved strand separates away from its complementarystrand leaving the chemiluminescent label exposed. Thereafter, whenexposed to hydrolysing conditions the chemiluminescent label isdestroyed. It therefore follows that this assay method is equallyeffective at assaying for the activity or presence of an enediyne.

In a modification of the Scheme shown in FIG. 10 the chemiluminescentlabel may not be attached to the duplex but instead provided on aseparate oligonucleotide which is complementary, at least in part, to aportion of the duplex whereby in the presence of the enzyme or theenediyne the duplex is cleaved and the oligonucleotide can bind to itscomplementary portion of the duplex. Thus, in this variation of SchemeG, the binding of the labelled oligonucleotide to a fragment of theduplex is indicative of the presence of the enzyme or the enediyne andproportional to the activity thereof.

Scheme H (FIG. 10)

This scheme is designed for monitoring the activity of intergraseenzymes.

The substrate for this enzyme consists of two oligonucleotides. Theycontain inter and intra complementary sequences which are able tohybridise to produce the secondary structure depicted. Intergrasecleaves and ligates this structure such that a chemiluminescent (AE)labelled strand is incorporated into the larger of the twooligonucleotides. Upon exposure to elevated temperature theunincorporated, or smaller, oligonucleotide melts off. The largeroligonucleotide is then exposed to hydrolysing conditions and thechemiluminescent label, ligated into the long strand by the intergrase,can take shelter in the coil of the double stranded nucleic acid and soresist degradation.

In this scheme the signal of the chemiluminescent label is directlyproportional to the activity of the intergrase enzyme. Thus, the moreactive the enzyme, the more chemiluminescent label is incorporated intoduplex and so the more it can be protected from the degradative effectsof hydrolysis.

Scheme I (FIG. 11)

This scheme is designed for monitoring the activity of topoisomeraseenzymes.

A duplex nucleic acid with a 5′ duplex extension is first manufactured.One of the strands of this duplex further includes a specific cleavagesite for the enzyme topoisomerase. If topoisomerase is present oractive, then it acts upon the duplex, at the cleavage site, to produce aduplex with an extended 5′ extension.

A chemiluminescent labelled oligonucleotide, complementary to saidextended 5′ extension is then added to the assay. The topoisomerase thenligates this oligonucleotide thus producing a chemiluminescent labelledduplex.

Upon exposure to degradation conditions, by way of hydrolysis, saidchemiluminescent label is protected from hydrolysis in the coil of theduplex. In contrast, any unligated oligonucleotide is destroyed.

In this assay the signal intensity of the chemiluminescent label isdirectly proportional to the activity of the topoisomerase enzyme. Thus,the amount of signal increases as the enzyme acts to incorporate theoligonucleotide label into the extended duplex.

DETAILED DESCRIPTION OF THE INVENTION

Indirect Ligase Activity Assay Based on Scheme ‘A’

Here a first oligonucleotide strand is synthesised which comprises asequence of nucleotides complementary to a second (target) strandpresent in nicked or unligated form. The unligated second strandrepresents at least part of a strand capable of acting as a ligaseenzyme substrate which is converted to a repaired or ligated strand bythe action of the enzyme. The strand is “nicked” preferably at aposition where the ratio of the relative lengths of the two componentsof the unligated strand does not exceed four. The possible range ofpositions of the nick is constrained by the overall length of the nickedstrand. The third oligonucleotide strand has a nucleotide strandidentical to the said first oligonucleotide strand but which furthercomprises a “linker” moiety to which can be attached a chemiluminescentor fluorescent emitter molecule. The synthesis of such labelledoligonucleotides is well-established. Preferably the first and thirdoligonucleotide strands comprise nucleotide strands of between 10 and 60bases, more preferably between 20 and 40 bases. Preferably the emittermolecule is a chemiluminescent molecule, more preferably the emittermolecule is a chemiluminescent acridinium salt.

A suitable ligase substrate is prepared by admixture of said first andsecond strands such that a nicked duplex is produced similar to that instep (i) of Scheme A. In practice the second strand comprises twoshorter strands one of which is phosphorylated at its free 5′-end by asuitable method for example by using T4 polynucleotide kinase.Preferably 10-100 nmol of each strand is hybridised in suitable buffer,preferably lithium succinate 1-100 mmol/l, 0.1-1 ml for preferably 0.5-2hours at 60° C. A suitable amount of this substrate is then admixed withthe desired amount of enzyme and the reaction allowed to proceed for anappropriate period of time under the usual conditions.

The labelled third oligonucleotide strand is dissolved in a buffermedium which is compatible with the labelled strand in terms of allowingit to hybridise to the second oligonucleotide strand and in terms ofmaintaining the stability of the reagents during the hybridisationreaction. The formulation of such buffers is established in this field.Typically the buffer ions consist of organic and/or inorganic saltspreferably at concentrations in the range 1 to 100 mmol/l and thesolutions may contain other solutes such as surfactants and/orpreservatives and possess pH values preferably of seven or less. Theamount of labelled oligonucleotide used depends on the sensitivity ofdetection of the label and the sensitivity of detection of target strandrequired in the assay. It is known that, typically, chemiluminescenceemission can be more sensitively detected than conventional fluorescenceemission and that therefore fluorescent probes may be inappropriatewhere very high sensitivity of detection is required. The amount oflabelled oligonucleotide used for an individual determination maytypically lie in the range 10⁻¹⁸ to 10⁻⁹ mol, more preferably 10⁻¹⁵ to10⁻¹² mol. This may be contained in a volume of buffer in the range 1microlitre to 1 millilitre, though this could be less than 1 microlitrein certain situations.

The solution of labelled probe is admixed with the analytical sample ina suitable reaction vessel such as a discrete test tube, or part of anarray of reaction vessels such as a 96, 384 or 1536 well microtitreplate. Alternatively it is known that many analysis procedures make useof solid-phase systems involving the use of immobilised microarrays andit will be appreciated that the means described herein can be extendedto such systems in parallel to the manner in which conventional labelledprobe assays have been used.

The hybridisation reaction is allowed to proceed at a temperaturetypically in the range 4-80° C., more preferably in the range 30-60° C.for a period of time typically in the range 1 minute to 240 minutes,more typically 5 minutes to 30 minutes.

Following the first incubation there a degradation stage in which thereis added to the reaction mixture a degradation reagent capable ofcausing one or more bonds in the label moiety to dissociate in such amanner that where the label is part of an intact duplex it is protectedfrom the said dissociative process. The dissociative processes generallyalso require the use of elevated temperatures. The degradation reagentmay be a buffer solution with a pH greater than 7 which is capable ofbringing about hydrolysis of the label moiety. The invention is notlimited to the use of hydrolysis and extends to other ways ofselectively inhibiting the ability of the emitter label to produce lightdepending on whether the emitter label is part of an intact duplex ornot. Examples of other ways of performing such selective dissociationreactions are disclosed in the literature (Ishikawa and Kato). In thistechnique, the intensity of chemiluminescence emission is proportionalto the ratio of ligated to unligated nucleic acid.

In the above assay to determine ligase activity with a DNA enzymesubstrate, the hybridisation reaction is preceded by a reaction step inwhich the enzyme, if present, acts to cause changes in the structure ofa nucleic acid. In the case of ligase, this involves repairing “nicks”in the nucleic acid. The nucleic acid is then heated to denature orseparate the hybridised strands and subsequently cooled to allow thestrands to rehybridise. Where it is desired to determine whether or nota compound or mixture of compounds is capable of inhibiting oractivating the enzyme activity, the said enzyme is exposed to the saidcompound or mixture of compounds and its activity, or lack thereof, asassayed is compared with the assayed enzyme activity of enzyme not soexposed. In a similar manner the activity of any chemical or physicalsystem causing the conversion of “substrate” to product can bedetermined as can the activity of inhibitors or activators thereof.

Direct Ligase Activity Assay—Based on Scheme C

A “contrived” enzyme substrate is produced comprising a double-strandedoligonucleotide strand having between 20 and 60 base pairs, and one ofthe strands possessing at least one “nick” such that the nicked strandsare unligated. Furthermore, one of the strands of the nicked strandpossesses a linker and hydrolysable chemiluminescent label as describedabove. The substrate is used in an assay for ligase enzyme activity inwhich the substrate and enzyme are admixed under conditions appropriatefor the particular ligase enzyme being used, and which ensure that thedouble-stranded substrate does not dissociate into single strands duringthe enzyme reaction.

Subsequent to the exposure of the substrate to the enzyme, the reactionmixture is exposed to an elevated temperature typically in the range 35to 75° C., more preferably in the range 45 to 65° C. in order tohydrolyse any unprotected chemiluminescent label. Such hydrolysis isalso facilitated where necessary by prior addition of an appropriatebuffer solution to raise the pH of the reaction mixture preferablywithin the range 7 to 9.

Following the selective hydrolysis step, the reaction mixture is placedin a luminometer where the chemiluminescence emission is initiated andmeasured. The method of initiation of the chemiluminescent reaction isdependent on the particular chemiluminescent label being used, suchmethods being known to those skilled in the art. In one example wherethe label is a chemiluminescent acridinium salt, the initiation istypically effected by the addition of hydrogen peroxide and alkali. Awide range of suitable instruments for chemiluminescence detection iscommercially available.

Whilst the procedures described above relate to monitoring ligaseactivity, they may be used for any enzyme which facilitates theinterconversion of ligated and unligated nucleic acids. These procedureswill start with, or be preceded by, a method in which the enzyme beingtested is mixed with the nucleic acid substrate under conditions and inthe presence of any co-factors necessary for the reaction to proceed.Also at this point, or earlier, there may be added a substance to beinvestigated as to its possible effect on the activity of the saidenzyme.

The reaction conditions compatible with the activity of a given enzymeare well established in the literature and can be applied to theteachings herein. Moreover the general procedures which represent thepreferred modes for bringing about the interactions between enzymes andinhibitors are well-known. Accordingly, the techniques disclosed hereinmay be adapted to allow for the study of any chemical or physicalvariable affecting the activity of the enzymes described herein.

Ultimately, the intensity of chemiluminescence is proportional (eitherdirectly or indirectly depending on the methodology) to the ratio of theconcentration of ligated to unligated strand and as such is anindication or measure of the activity, inactivity or inhibition ofactivity of the enzyme present in the system.

The methods described can be applied as a means of determining theactivity of a range of enzymes which are responsible for themodification of nucleic acid and which involve ligation and/or cleavageas part of their overall function. In this situation, the temperature atwhich the hydrolysis procedure is carried out needs appropriateselection since it must also permit unligated duplex to melt and yetallow ligated duplex to remain intact and thus facilitate hybridisationprotection. Appropriate temperatures will be different for differentstrands and an empirical approach is required to optimise thistemperature for a given strand.

Similar experimental protocols may be used for the assay of the activityof helicase enzymes or inhibitors thereof except that in these cases thelabelled oligonucleotide strand is designed such that it is capable ofbinding to “unwound” genetic material that constitutes the product ofthe respective enzyme activity but incapable of binding to substrate asrepresented by a nucleic acid duplex. Lack of enzyme activity as occursupon enzyme inhibition by a chemical compound or mixture thereof resultsin the absence of accessible target for hybridisation of the labelledoligonucleotide strand.

Further, as set out in Scheme D, a helicase assay may utilise a“contrived substrate” in which one of the strands of the substrateduplex is itself labelled such that the properties of the label aredifferent when the duplex has been “unwound” by the enzyme. Thecontrived substrate duplex may be labelled with e.g. an acridinium esterwhose rate of hydrolysis is increased when that part of the nucleic acidstrand to which it is linked is separated from its complementary strandby the action of helicase. As described above, the physical/chemicalconditions are then altered to selectively hydrolyse the acridinium saltpresent in the product of the helicase reaction, whilst leavingsubstantially unaffected that which is present in the form of unreactedsubstrate. In this case the intensity of chemiluminescence is inverselyproportional to enzyme activity.

Similar experimental protocols may be used for the assay of the activityof integrase and transposase enzymes or inhibitors thereof. Herelabelled oligonucleotides may be used that are capable of hybridising tothe product nucleic acid strand (i.e. that following enzyme activity)but not the unmodified substrate nucleic acid strand, or vice versa.

It will be appreciated that if the substrate or product to be bound tothe labelled oligonucleotide strand exists as a duplex then it may benecessary to bring about dissociation of the said duplex beforehybridisation with the oligonucleotide probe can take place. Variousways of bringing about such dissociation are well-established in theart.

The following examples are illustrative of the principles, withoutlimitation as to the application, of the teachings embodied herein.

EXAMPLE 1

1. DNA Ligase Assay Using Hybridisation Protection of a ChemiluminescentAcridinium Ester Labelled Oligonucleotide Strand.

Three oligonucleotides were prepared using established methods. Thestrands of these were as follows: (i) 5′-GGC CTC TTC GCT ATT ACG CCAGCT-3′ (ii) 3′-CCG GAG AAG CGA-5′ (iii) 3′-TAA TGC GGT CGA-5′

Also prepared by published methods was a chemiluminescent derivative of(i) as follows (* represents the position of the chemiluminescent label)(iv) 5′-GGC CTC TTC GCT*ATT ACG CCA GCT-3′

The free 5′-end of (ii) was phosphorylated by established methods. Thephosphorylation ensures that the strands are nicked. Stock duplex wasformed by hybridising the phosphorylated (ii) with equimolar amounts of(i) and (iii) for one hour at 60° C. in lithium succinate buffer.Investigations of ligase activity were performed using mixtures of theduplex (6 pmol) and T4 DNA ligase (80 units) admixed with putativeinhibitors if required.

The reaction product was analysed for ligated product as follows:

Samples of the ligase product reaction mixture were diluted 1000-fold intris buffer (0.01 mol/l, pH 8.3) for analysis by hybridisationprotection assay. 100 ul of the dilutions were added to labelled probe(iv) (50 fmol) diluted in reaction buffer (125 mmol/l lithium hydroxide,95 mmol/l succinic acid, 1.5 mmol/l EGTA, 1.5 mmol/l EDTA, 8.5% lithiumlauryl sulphate, pH 5.2) in 500 ul microcentrifuge tubes. The tubes wereincubated at 95° C. for 5 minutes followed by an incubation at 60° C.for 30 minutes. The tubes were cooled to 4° C. and 100 ul of thecontents of each tube transferred to corresponding 12×75 mm polystyrenetest tubes. Hydrolysis reagent (190 mmol/l sodium borate, 5% TritonX-100, pH 7.6)(300 ul) was then added and the tubes incubated at 60° C.for 10 minutes. The tubes were placed in an ice bath for one minute andthen placed in a luminometer (Stratec Biomedical Systems, Pforzheim,Germany) programmed to sequentially inject 200 ul each of DetectionReagents 1 and 11 (Gen Probe Inc., San Diego, USA) with a read time of 5seconds.

FIG. 9 shows the effect on the enzyme of a known ligase inhibitor(ethylene diamine tetra-acetic acid, EDTA).

2. DNA Ligase Assay Using Hybridisation Protection of a ChemiluminescentAcridinium Ester Labelled Duplex Substrate.

Oligonucleotides (ii), (iii) and (iv) from Example 1 were hybridised inthe same way as previously used for strands (i), (ii) and (iii). Thestock labelled duplex was then used directly in the ligase assay.

Hydrolysis reagent was added as before and chemiluminescencemeasurements carried out as described above.

EXAMPLE 2

Reverse Transcriptase (RT): Inhibition of by Di-Deoxy ThymidineTriphosphate (ddTTP).

Assay template was a pre-primed 81 nt DNA (non-sense) oligonucleotideconsisting of sequential primer, T7 viral DNA dependent RNA polymerasepromoter and reporter sequences. RT dependent extension of a shortpre-hybridised sense strand primer yields double strandedpromoter/reporter and enables RT regulated T7 RNA polymerase generationof report mRNA transcript. Template was incubated in buffer containingrTNPs (2 mM), dTNPs (0.1 mM), avian myeloblastosis virus RT, 17 RNApolymerase and serial dilutions of ddTTP. Reporter mRNA product was thenmeasured by HPA (Hybridisation Protection Assay). Briefly,oligonucleotides complementary to the substrate strand, or itscomplementary counterpart, where hybridised to the corresponding strandof DNA after exposure to a melt temperature.

Hydrolysis reagent was added as before and chemiluminescencemeasurements carried out as described above.

EXAMPLE 3

DNA Helicase: Time Course of Strand Separation at Three Enzyme:Substrate Ratios.

AE labelled double stranded substrate was incubated in the presence ofenzyme. Unseparated substrate confers hybridisation protection to AE andthus signal intensity is inversely proportional to enzyme activity.

EXAMPLE 4

T7 DNA Dependent RNA Polymerase Generation of mRNA: Inhibition DoseResponse Using EDTA.

Template was PCR generated linearised DNA containing the 17 RNApolymerase promoter and coding for a 295 nt mRNA transcript includingreporter target sequence. Template plus enzyme were incubated in serialdilutions of EDTA as model inhibitor. Reporter mRNA product was thenmeasured by hybridisation protection assay, HPA. Briefly, labelledoligonucleotides complementary to the newly formed strand werehibridised to same. Hydrolysis reagent was added as before andchemiluminescence measurements carried out as described above.

EXAMPLE 5

E coli RNA Polymerase: Inhibition by Rifampicin.

Stock template was constructed from a 64 nt synthetic oligonucleotidecoding sequentially (3′-5′ non-sense) for consensus sequence RNApolymerase promoter and reporter mRNA transcript. A short sense strandprimer was annealed at the 3′ terminus and the complete duplex extendedusing Klenow DNA polymerase. Template was incubated in assay bufferusing E coli RNA polymerase holoenzyme with serial dilutions ofinhibitor in DMSO. Reporter mRNA product was then measured byhybridisation protection assay HPA. Briefly, labelled oligonucleotideswere hybridised to the newly formed strand. Hydrolysis reagent was addedas before and chemiluminescence measurements carried out as describedabove.

1. A method for determining the activity of a microbial or viral enzymecapable of altering the structure of a nucleic acid from a first stateto a second state comprising the steps of: (a) providing in a testsample; (i) said enzyme selected from the group consisting of: a ligase,helicase, polymerase, reverse transcriptase, primase, nuclease,integrase, topoisomerase, transposase and gyrase: (ii) said nucleicacid; and, optionally, (iii) one or more oligonucleotides complementary,at least in part, to said nucleic acid when in said first or secondstate; wherein either, or both, of said oligonucleotide or said nucleicacid comprises a label capable of providing an output signal, andfurther wherein the stability of said label against degradation isdifferent depending upon whether said nucleic acid is in said first orsecond state; (b) exposing said test sample to degradation conditions;(c) detecting said output signal and thereby determining whether saidnucleic acid is, at least predominantly, in said first or second state;and (d) determining the activity of said enzyme. 2-3. (canceled)
 4. Amethod according to claim 1 wherein said nucleic acid is DNA or RNA. 5.A method according to claim 1 wherein said nucleic acid is either singlestranded or multi-stranded.
 6. A method according to claim 1 or 4wherein said enzyme is a ligase, said nucleic acid in its first state ismulti-stranded, and step (a) further comprises subjecting said sample toa temperature that causes said multi-stranded nucleic acid to separateinto single strands and so enables any ligated nucleic acid strand, orits complementary strand, to hybridise with said oligonucleotide.
 7. Amethod according to claim 1 or 4 wherein said enzyme is a ligase, saidoligonucleotide is omitted from said test sample and said nucleic acid,which is multi-stranded, comprises said label, and step (a) furthercomprises subjecting said sample to a temperature that causes anyunligated nucleic acid, at least partially, to separate into singlestrands.
 8. A method according to claim 6 wherein said temperature isselected so that unligated nucleic acid separates but ligated nucleicacid does not separate.
 9. A method according to claim 6, 7 or 8 whereinsaid nucleic acid comprises an interrupted strand made up of at leasttwo unligated portions capable of being ligated by said enzyme.
 10. Amethod according to claim 1 or 4 wherein said enzyme is a helicase, saidnucleic acid in its first state is multi-stranded, and step (a) furthercomprises subjecting said sample to an environment which allows, atleast partial, unwinding of said nucleic acid.
 11. A method according toclaim 10 wherein said oligonucleotide is omitted from said test sampleand said nucleic acid comprises said label.
 12. A method according toclaim 1 wherein said enzyme is a polymerase, said nucleic acid is in theform of oligonucleotides and/or nucleotides, and step (a) furthercomprises subjecting said sample to an environment which allows saidoligonucleotides and/or said nucleotides to join to form a strandwhereby said complementary oligonucleotide can bind thereto.
 13. Amethod according to claim 1 wherein said enzyme is a reversetranscriptase or primase, said nucleic acid is in the form of nucleosidetriphosphates, and step (a) further includes comprises subjecting saidtest sample to an environment which allows said nucleoside triphosphatesto join to form a strand whereby said complementary oligonucleotide canbind thereto.
 14. A method according to claim 12 or 13 wherein saidsample further comprises a nucleic acid template.
 15. A method accordingto claim 14 wherein said template is single stranded.
 16. A methodaccording to claim 1 wherein said enzyme is a nuclease, saidoligonucleotide is omitted from said sample and said nucleic acid, whichis multi-stranded and comprises a site specific cleavage point,comprises said label, and step (a) further comprises subjecting saidsample to a temperature that causes any cleaved nucleic acid to separateinto single strands.
 17. A method according to claim 1 wherein saidenzyme is a nuclease, said nucleic acid is multi-stranded and comprisesa site specific cleavage points and step (a) further comprisessubjecting said sample to a temperature that causes any cleaved nucleicacid to separate into single strands whereby said complementaryoligonucleotide can bind to at least a selected one of said strands.18-19. (canceled)
 20. A method according to claim 16 or 17 wherein saidtemperature is selected so that said cleaved nucleic acid separates butuncleaved nucleic acid does not separate.
 21. A method according toclaim 1 or 4 wherein said enzyme is an integrase, said oligonucleotideis omitted from said sample and said nucleic acid comprises at least twooligonucleotides containing inter and intra complementary sequences, andfurther wherein one of said sequences comprises said labels and step (a)further comprises subjecting said sample to a temperature that causesany unincorporated oligonucleotide to separate or melt away.
 22. Amethod according to claim 7, 16 or 21 wherein said label is locatedremotely from the site at which said enzyme is active whereby said labelcannot interfere with the activity of said enzyme.
 23. A methodaccording to claim 1 or 4 wherein said enzyme is a topoisomerase, saidnucleic acid in its first state comprises a duplex with a 5′ or 3′extensions and said oligonucleotide is a religation strand, that is astrand that is complementary to the 5′ or 3′ extension produced by theaction of said enzyme.
 24. A method according to any one of thepreceding claims wherein detecting said output signal comprises the useof one or more of colourimetric, fluorimetric or chemiluminescent means.25. A method according to any one of the preceding claims wherein saidlabel is a fluorescent or chemiluminescent molecule.
 26. A methodaccording to claim 25 wherein said label is an acridinium salt.
 27. Amethod according to any one of the preceding claims wherein the activityof more than one enzyme is determined and said method comprises thesteps of: (a) providing in a test sample: (i) a plurality of enzymes;(ii) nucleic acid for said enzymes; and, optionally, (iii) one or moreoligonucleotides complementary, at least in part, to said nucleicacid(s) when in said first or second state; wherein either, or both, ofsaid oligonucleotide or said nucleic acid comprises a plurality oflabels, each capable of providing an output signal which signal isstable against the effects of degradation depending upon whether saidnucleic acid is in said first or second state, and further wherein theoutput signal of each label differs whereby the activity of each enzymecan be distinguished; (b) exposing said test sample to degradationconditions; (c) detecting said output signals of each label and therebydetermining whether said nucleic acid(s) is, at least predominantly, insaid first or second states; and (d) determining the activity of each ofsaid enzymes. 28-32. (canceled)
 33. A method for screening an agent formodulatory activity in relation to a microbial or viral enzyme capableof altering the structure of a nucleic acid from a first state to asecond state comprising the steps of: (a) providing in a test sample:(i) said enzyme selected from the group consisting of: a ligase,helicase, polymerase, reverse transcriptase, primase nuclease,integrase, topoisomerase, transposase and gyrase; (ii) said nucleicacid; (iii) an agent to be tested; and, optionally, (iv) at least oneoligonucleotide complementary, at least in part, to said nucleic acidwhen in said first or second state; wherein either, or both, of saidoligonucleotide and or said nucleic acid comprises a label capable ofproviding an output signal, and further wherein the stability of saidlabel against degradation is different depending on whether said nucleicacid is in said first or second state; (b) exposing said test sample todegradation conditions; (c) detecting said output signal and therebydetermining whether said nucleic acid is, at least predominantly, insaid first or second state; and (d) determining the activity of saidenzyme and thus the modulatory activity of said agent. 34-37. (canceled)38. A substrate nucleic acid for use in determining the activity of apredetermined microbial or viral enzyme selected from the groupconsisting of a ligase, helicase, polymerase, reverse transcriptase,primase, nuclease, integrase, topoisomerase, transposase and gyrasecomprising a complex of a nucleic acid and a label, said nucleic acidbeing capable of being acted upon by said enzyme whereby, on said enzymebeing active, the said substrate nucleic acid changes from a first stateto a second state thereby affecting the stability of said label againstdegradation. 39-44. (canceled)